Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice

The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and...

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Main Authors:	Mikami, Masafumi, Toki, Seiichi, Endo, Masaki
Format:	Online
Language:	English
Published:	Springer Netherlands 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523696/

id	pubmed-4523696
recordtype	oai_dc
spelling	pubmed-45236962015-08-06 Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice Mikami, Masafumi Toki, Seiichi Endo, Masaki Article The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants. Springer Netherlands 2015-07-19 2015 /pmc/articles/PMC4523696/ /pubmed/26188471 http://dx.doi.org/10.1007/s11103-015-0342-x Text en © The Author(s) 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Mikami, Masafumi Toki, Seiichi Endo, Masaki
spellingShingle	Mikami, Masafumi Toki, Seiichi Endo, Masaki Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
author_facet	Mikami, Masafumi Toki, Seiichi Endo, Masaki
author_sort	Mikami, Masafumi
title	Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
title_short	Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
title_full	Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
title_fullStr	Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
title_full_unstemmed	Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice
title_sort	comparison of crispr/cas9 expression constructs for efficient targeted mutagenesis in rice
description	The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants.
publisher	Springer Netherlands
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523696/
_version_	1613255361341423616

Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice

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